The cap-snatching endonuclease of influenza virus polymerase resides in the PA subunit
The cap-snatching endonuclease of influenza virus polymerase resides in the PA subunit
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DOI：
10.1038/nature07745
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发表时间：
2009-04-16
期刊：
影响因子：
64.8
通讯作者：
Ruigrok, Rob W. H.
中科院分区：
文献类型：
--
作者：
Dias, Alexandre;Bouvier, Denis;Ruigrok, Rob W. H.
流感病毒聚合酶是由PA、PB 1和PB 2三个亚基组成的异源三聚体，负责病毒RNA基因组的八个独立片段在感染细胞核中的复制和转录。聚合酶通过独特的“夺帽”机制，使用源自细胞转录物的短帽引物合成病毒信使RNA（1）。PB 2亚基结合宿主前体mRNA的59个帽（2-4），其随后在10-13个核苷酸后被病毒核酸内切酶切割，迄今为止被认为存在于PB 2（参考文献5）或PB 1（参考文献2）亚基中。在这里，我们描述的生化和结构的研究表明，氨基末端的PA亚基的209个残基含有核酸内切酶活性位点。我们表明，该结构域具有固有的RNA和DNA内切核酸酶活性，该活性被锰离子强烈激活，与完整三聚体聚合酶的内切核酸酶活性的观察结果相匹配（6，7）。此外，该活性被2，4-二氧代-4-苯基丁酸抑制，2，4-二氧代-4-苯基丁酸是已知的流感内切核酸酶抑制剂（8）。该结构域的晶体结构揭示了一个结构核心，非常类似于分解酶和II型限制性内切核酸酶。活性位点包括组氨酸和一簇三个酸性残基，在所有流感病毒中保守，其结合两个锰离子的配置类似于其他两种金属依赖性核酸内切酶。先前已显示两个活性位点残基在突变时特异性消除聚合酶核酸内切酶活性（9）。这些结果将有助于优化核酸内切酶抑制剂（10-12）作为潜在的新的抗流感药物。
The influenza virus polymerase, a heterotrimer composed of three subunits, PA, PB1 and PB2, is responsible for replication and transcription of the eight separate segments of the viral RNA genome in the nuclei of infected cells. The polymerase synthesizes viral messenger RNAs using short capped primers derived from cellular transcripts by a unique 'cap-snatching' mechanism(1). The PB2 subunit binds the 59 cap of host pre-mRNAs(2-4), which are subsequently cleaved after 10-13 nucleotides by the viral endonuclease, hitherto thought to reside in the PB2 (ref. 5) or PB1 (ref. 2) subunits. Here we describe biochemical and structural studies showing that the amino-terminal 209 residues of the PA subunit contain the endonuclease active site. We show that this domain has intrinsic RNA and DNA endonuclease activity that is strongly activated by manganese ions, matching observations reported for the endonuclease activity of the intact trimeric polymerase(6,7). Furthermore, this activity is inhibited by 2,4-dioxo-4-phenylbutanoic acid, a known inhibitor of the influenza endonuclease(8). The crystal structure of the domain reveals a structural core closely resembling resolvases and type II restriction endonucleases. The active site comprises a histidine and a cluster of three acidic residues, conserved in all influenza viruses, which bind two manganese ions in a configuration similar to other two-metal-dependent endonucleases. Two active site residues have previously been shown to specifically eliminate the polymerase endonuclease activity when mutated(9). These results will facilitate the optimisation of endonuclease inhibitors(10-12) as potential new anti-influenza drugs.